Field of the Invention
The present invention provides a novel method to produce Rebaudioside D (RD) from Rebaudioside B (RB) by using cutinase to catalyze the conversion under stepwise temperature changes, which relates to the field of biosynthesis of organic compounds.
Description of the Related Art
Rebaudioside D, (4R)-13-[[2-O-(β-D-Glucopyranosyl)-3-O-(β-D-glucopyranosyl)-β-D-glucopyranosyl]oxy]kaur-16-en-18-oic acid 2-O-(β-D-glucopyranosyl)-β-D-glucopyranosyl ester, is a sweetener which exists in trace amounts in stevia rebaudiana. It has a texture and taste more similar to saccharose than Rebaudioside A (RA), which is currently used and could be obtained in large amounts. RD is usually used as a flour enhancer for RA as well as other sweeteners. RD was certified by GRAS as a trace substance in stevia rebaudiana in September 2010, and it also got certified as high-purity substance by GRAS in April 2015.
The main method to produce RD is phytoextraction, while chemical synthesis of RD has been rarely reported. The structure of RD is similar to RB, 13-O-[β-D-glucopyranosyl(1→2)-(β-D-glucopyranosyl(1→3))-β-D-glucopyranosyl]-ent-13-hydroxy-kaur-16-en-19-oic acid. RB is a steviol glycoside that could be made from RD by specific hydrolysis of the sophorosemoiety on C19 of RD. It is also a sweetener that exists in trace amounts in stevia rebaudiana (generally less than 0.1%). RB gives a bitter aftertaste and it can be produced by enzymatic or alkalic hydrolysis of glucose on C19 of RA. Hence, from the structural point of view, RD can be produced by esterification of sophorose with RB.
The esterification could be catalyzed by enzyme or acid. The acidic esterification is easy to conduct, in which the hydroxyl groups in RB should be protected to avoid the esterification between RB molecules. Acetylation is a well-known method to protect the hydroxyl group, and is used to protect the saccharide hydroxyl in sucralose synthesis. The enzymatic esterification is more gentle and specific than chemocatalysis. If esterification between RB and sophorose can be conducted specifically, the protection and deprotection step can be avoided. The enzymatic synthesis of glycolipid is commonly reported. There are more than 10 types of popular commercial lipases, and the most popular lipase is Novazym435. For example, Novozym435 catalyzes the reaction between lauric acid and sucrose in tertiary butanol to produce sucrose monolaurate and sucrose dilaurate. As another example, Novazym435 can catalyze the synthesis of trehalose. Enzymes from different sources have different substrate selectivity for the chain length of the acylating reagents. Enzymes from multiple sources of bacteria and fungus could catalyze glucose monoester reaction, for example, the pseudomonas and fungus's enzymatic activity with the carbon source in culture media is outstanding. But RB is an acid with 3 glucose residues, which is different with the aliphatic acids mentioned above. After screening more than twenty commercial lipases, none of the lipases can catalyze the sophorose esterification onto RB. This may be due to the solubility issue of RB and the steric hindrance of hydroxyl on RD. In addition, the solvent of RB always has a remarkable inhibitory effect to lipases, which made it difficult to perform enzymatic esterification by lipases.